The invention relates to a solid-phase support for a component adapted to participate in a reaction in a liquid phase
Solid-phase granules are finding increasing applications in liquid-phase reactions, for the purpose of analysis (e.g. solid-phase immunological tests) and also in manufacturing processes (e.g. immobilised enzyme or cell systems for industrial fermentation).
In most immunological tests, a tracer is used for measuring the distribution of antigen-antibody complexes and antigens or antibodies alone in the reversible-bond antigen-antibody reaction, when the distribution between the bonded complexes and the free parts is proportional to the initial concentration of antigens-antibodies before the reaction
Provided the immunological test tracer is not modified by the bonding reaction (as in homogeneous tests, e.g. EMIT.RTM. Syva, U.S.A.), the determination process is facilitated by separating the bonded complexes from the free parts of the test (as in a heterogeneous test). This result is often obtained by fixing a test component to a solid phase which is physically separated from the liquid phase after the incubation reaction A number of variants of heterogeneous tests is known and described in the literature.
A number of different types of solid phases intended for heterogeneous bond tests is commercially available. The available range extends from single-support systems such as coated balls or tubes, to microcrystalline cellulose or fine-particle silica. The choice of solid phase for a given test will depend on a number of factors, inter alia the ability to be associated with a bonding agent (relative to the total available surface area) and the convenience of use.
The disadvantage of these solid phases are that they have to be dispersed in the liquid phase in order to bring about a complete reaction (at equilibrium) with the reagent or reagents in solution, and they must then be separated from the aqueous phase before measuring the result of the reaction.
A solid-phase support for immunological tests therefore has to satisfy two contradictory requirements. To obtain optimum dispersion of the solid phase resulting in an efficient reaction and consequently a sensitive test, it is necessary for the solid phase to have the largest possible surface area in a given volume, and this can be obtained with fine granules. On the other hand, separation of the solid phase will be better in proportion as the granules are larger. The adopted methods usually try to make a compromise between these two contradictory requirements and discover the most efficient methods of mixing and separation.
For the purpose of mixing, for example, it has been proposed to vibrate the reaction tubes around a transverse axis at the centers of the respective tubes, or intermittent vortex mixing, or intermittent agitation or use of fine granules. For the purpose of separation, use has been made of centrifuging and repeated washing of the solid phase (with intermediate mixing), or of a saccharose gradient, or ferromagnetic particles, plastics balls, etc.
Ferro-magnetic particles can easily be separated from the liquid by using a magnetic field. Although these particles are relatively heavy, they remain fairly well in suspended in the liquid phase, provided they are very finely divided. These particles, however, are difficult to re-suspend in the liquid phase.
Irrespective of the nature of the solid-phase supports in the form of particles or the aforementioned granules for suspending in the liquid phase, it is necessary to use two different techniques for measuring and separating the particles or granules. Frequently, these techniques are difficult to operate in an automatic analysis installation. The mixing operation, for example, which should be performed without risk of contamination and without removing the product, should preferably avoid any contact with the liquid for analysis. Agitation techniques, particularly with very small volumes of liquid, are not always efficient or reproducible, owing to the surface tension inter alia, against the vessel walls. Separation by centrifuging usually necessitates transferring the analysis tube to a centrifuging rotor, which increases the complexity of the installation and the process of analysis and also increases the cost and the amount of manipulation.
An intermediately-sized solid phase is a compromise between the two extremes, i.e. finely divided particles and a single support. An aforementioned intermediate-size solid phase has an adequate bonding-agent capacity and, if its density is substantially equal to that of the liquid phase, it can remain in suspension during the incubation phase of the main reaction.
Patent Application EP-A-O 123 403 relates to intermediate-sized particles which are slowly separated by gravity. The disadvantage of separation during incubation is avoided by substantially increasing the density of the liquid phase during incubation (e.g. by adding saccharose). However, this method has certain disadvantages. For example, addition of an agent for modifying the density mat influence the reaction as a result of chemical or physical effects on the bonding properties. Addition and dilution of the aforementioned agent in the liquid phase may affect the balance obtained and necessitate additional steps in the test.
Solid phases are also being increasingly used in sectors other than immunological analysis, e.g. in biological and biochemical reactors. Some cells and micro-organisms can be more efficiently cultivated on suitable surfaces such as 50-300 .mu.m microsupports, e.g. Cytodex.RTM. produced by Pharmacia (Sweden) and used for production of viruses, interferon and antibodies. Viruses and vaccines are produced in this manner in 100-1 000 litre reactors using solid-phase support particles.
Since micro-organisms grow and can undergo a number of undesirable secondary reactions, it is sometimes preferable to mass only those enzyme systems necessary for the desired mass reaction. These enzymes are soluble in the liquid phase and will therefore be washed in a continuous reactor. For this reason they are often immobilised on solid phases. Although the whole organisms or cells are more stable and they can synthesise their own enzymes and arrange the steric positioning of the reagents in the required reactions, enzymes are often used for practical reasons, i.e. for monitoring and for obtaining a pure product.
Cultivation of mammal cells is often the only appropriate means of producing mammal proteins, since mammal cells undergo a number of the post-translational modifications necessary for producing functional molecules. Co-ordination of mammal cells is difficult owing to the fragility of the cells, which are sensitive to conventional mixing methods such as mashing and stirring Since mammal cells grow relatively slowly, it is important to maintain aseptic conditions and avoid an accumulation of microbial products which may reduce the growth of cells.
Mammal cells are grown by two methods -- in free homogeneous suspension (under good cultivation conditions) and in immobilised form with perfusion by the liquid phase (e.g. in hollow-fibre reactors, fluidised beds or on spongy or ceramic matrices.)
Conventional mixing devices comprise mechanisms for moving, agitating or rotating the reaction vessel or conveying air bubbles through the reaction medium These mixing processes have disadvantages. Mashing of the liquid results in dislocation through shearing and the seal of the mashing shaft is a source for contaminating the reactor. Agitation and rotation of the reactor are not very efficient and cause contamination of the reactor walls. Mashing with air bubbles may result in the formation of foam and contamination of the reactor walls.
Perfusion reactors are being increasingly used for large-scale production of substances having a high added value, such as monoclonal antibodies. The disadvantage of hollow-fibre reactors is the pressure drop associated with perfusion through hollow fibres and the possibility of forcing pockets of isolated cells in a micro-environment. Consequently, some manufacturers still prefer to use suspension reactors in the form of a fluidised bed.
As in immunological tests, the main problems of using solid phases relate to the mixing of the solid phases with the reagents, to ensure a complete reaction and subsequent separation of the reaction products in the liquid phase. In large-scale production, mixing is also important in transferring the heat of reaction and distributing the gas in the liquid phase.